The differences in adult lifetime among various silkworm strains has been suggested that the adult lifetime may be genetically controlled. In this experiment, using J037 and Daizo strains we investigated genetic factors related to the adult lifetime of silkworm. We constructed the full-length cDNA library from the adult male of the J037 strain. A total of 2,688 clones were randomly selected, and we performed a differential display hybridization with cDNA probes generated from J037 and Daizo adult males. In conclusion, 193 clones were identified as differential expressed genes, and 154 unique genes were generated after the assembly of 193 clones. Of the 154 unique genes, the most abundant genes were cytochrome oxidase subunit-1 gene(9 times) and unknown(clone ID; 1-50) gene(5 times). The functional groups of these unique genes with matches in the AmiGo database were constructed according to their putative molecular functions. Among thirteen functional categories, the largest group was unclassified protein(24%). In addition, We analyzed the nucleotide and deduced amino acid sequences of the most highly occurred gene(1-50, EF434397), which consisted of 240 amino acids. However, it is confirmed yet that these genes really have an affected on the silkworms longevity.

Insect cuticular melanization is regulated by the prophenoloxidase (proPO)- activating system, which is also involved in the innate immune reaction. Here, we demonstrate how the differentiation of the proPO-activating system is regulated toward a cuticular melanization or innate immunity function in silkworm (Bombyx mori) pupae. Our results indicate that the differential and spatial regulation of key components, such as the proPO-activating factor, tyrosine hydroxylase, and porPOs, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism that is mediated by the priming of the proPO system.

Antimicrobial peptides (AMPs) can be produced in mealworms, currently being used as animal feeds, by the infection of genetically engineered-entomopathogenic fungi. In this work, we integrated Bombyx mori (Bm) AMP, cecropin A to Beauveria bassiana ERL1170 by restriction enzyme-mediated integration method, which was confirmed by RT-PCR and an antibacterial activity assay. For the extracellular secretion of Bm cecropin A protein, the active domain of the cecropin A gene was tailed to the signal sequence of B. bassiana chitinase (Bbs). To exchange Bbs-cecropin A gene with egfp gene in pBARKS1-egfp, Bbs-cecropin A fragment was cut from pGEM-Bbs-cecropin A using XbaI/blunted and BamHI and ligated with cut pBARKS1-egfp using NcoI/blunted and BamHI, designated as pBARKS1-Bbscecropin A. After the transformation, transformants were grown on Czapek’s solution agar containing 600 μg ml-1PPT. Expression of Bm cecropin A was confirmed by RT-PCR. Strong clear zone was observed in the co-culture of the transformant D-6 and Bacillus subtilis on fourth strength Sabouraud dextrose agar 1 day after the culture at 25°C, whereas the wild type had no clear zone. This work suggests that Bm cecropin A can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry.

It is true that the proper environmental risk assessments for many GM insects almost have not been executed in Korean research situation. Therefore, we tested the environmental risk assessment of GM silkworms if there is any difference between GM and non-GM silkworms by three. First, we measured their mobility in the breeding environment conditions with food and without food. Secondly, we measured their viability at the Korean artificial extreme environmental conditions (temperature, humidity, food) after escaping from their breeding environments. We observed the egg productivity and the hatchability between non-GM silkworm and transgenic silkworms with four different pair experiments. The mobility of non-GM silkworms and GM silkworms statistically did not differ and the egg production and hatchability was not also different. The hatchability by couple of GM female silkworm and non-GM male silkworm was lower than by non-GM male and female couple. We observed their viability (High Temp., wet and with food: p=0.0434; High Temp., wet and without food p=0.0430; High Temp., dry and with food: p=0.0005; High Temp., dry, without food: p=0.0479) between the GM silkworm and non-GM silkworm, and there was statistically different. Relatively, the viability of GM silkworm was lower than non-GM silkworms. We could not exactly test for viability of silkworm in low temperature conditions because of their hibernating. Although there was any difference in viability and hatchability between GM silkworm and non-GM silkworm, the all ability of GM silkworm was lower than non-GM silkworm. Conclusively, risk of GM silkworm was lower than non-GM silkworm.

The anti-diabetes mechanism of silkworm Bombyx mori L. powder and extracts was found to inhibit the activity of α-glycosidase. The major functional component of silkworm powder was 1-deoxynojirimycin (1-DNJ), which exerts a blood glucose-lowering effect. In this study, we aimed to compare the effects of the supplements, including red ginseng extract on the functional components of silkworm. Fifty silkworm larvae were divided into the control group (Con, N=50), group A (A, artificial diet 95% and mulberry leaf powder 5%), group B (B, artificial diet 95% and mulberry powder 5%), group C (C, artificial diet 95% and Rubus coreanus remainders 5%), group D (D, artificial diet 95% and red ginseng extract 5%), and group E (E, artificial diet 95% and yeast powder (Saccharomyces cerevisiae). Body weights and length of silkworm larvae showed significant improvement in group A, D. In particular, the growth rate in group D (artificial diet 95% and red ginseng extract 5%) was larger than that of Con. In addition, the results showed that 1-DNJ concentration was significantly largest in group D. From these results, it is concluded that the addition of red ginseng extract may be effective for larval growth and 1-DNJ accumulation in silkworm rearing with an artificial diet.

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to meet a multi-parellel process. We have developed a novel recombinant bacmid, bEasyBm that enabling easy and fast generation of pure recombinant virus without any purification step. In the bEasyBm, attR recombination sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus early promoter. Therefore, only when the barnase gene was replaced to gene of interest, the bEasyBm could replicate in host insect cells. When the bEasyBm was transposed with pDualBac-EGFP and pDualBac-LUC respectively, there were no non-recombinant backgrounds were detected from unpurified BmEasy-EGFP or BmEasy-LUC stocks. In addition, the resulting recombinant virus, BmEasy-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, BmEGFP, which was constructed using bBmGOZA system. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established.

In arthropods, an immune challenge triggers a serine protease cascade that leads to the activation of prophenoloxidase (proPO)-activating factors (PPAFs), which are also called proPO-activating enzymes (PPAEs) or proteinases (PAPs). PPAFs are activated by cleavage between their clip and serine protease domains. Once activated, PPAFs convert proPO to phenoloxidase (PO), which then catalyzes the production of quinones to form melanin. In this study, we identified a Bombyx mori PPAF(BmPPAF) that involves in the pupal melanization. In the fat body, expression of BmPPAF was detected on day 1 to 3 of the pupal stage. RNA interference (RNAi)-mediated BmPPAF knock-down inhibited pupal melanization, resulting in the delay of pupal melanization. Based on these results, we concluded that BmPPAF is involved in the melanization of pupal stage in silkworm metamorphosis.

본 연구는 누에의 식엽성 곤충을 대체할 실험곤충으로서의 유용성과 자외선이 한국산 곤충병원성선충 (Heterorhabditis sp. 202 strain, Heterorhabditis sp. Gyeongsan strain, Steinernema sp. 223 strain, S. carpocapsae Pocheon strain, S. glaseri Dongrae strain, S. longicaudum Nonsan strain) 의 병원성과 생존에 미치는 영향을 알아보기 위하여 수행하였다. 누에에 대한 곤충병원성선충의 병원성을 조사한 결과 누에의 령기나 곤충병원성선충의 계통이나 종에 따라 상이한 병원성을 나타내었는데 5령충에 대해서는 Steinernematidae선충의 병원성이 높았고, 1령충에 대해서는 Heterorhabditidae선충의 병원성이 높았다. UV-C는 곤충병원성선충에 유해하여 노출 60분후에는 모든 선충이 치사되었다. UV-C 조사는 곤충병원성선충의 병원성에도 영향을 미쳐 노출 10분 후에도 누에나방 3령충에 대하여 6.7% 이하의 보정사충율을 나타내었다. 야외 뽕나무밭(UV량: 2.3mW/㎠)에서 곤충병원성선충, S. carpocapsae Pocheon strain을 엽면 살포 4시간 후 채취한 뽕나무 잎을 급상한 누에는 치사되지 않았다. 뽕잎에서 4.0mW/㎠의 UV 1시간 노출은 곤충병원성선충, S. carpocapsae Pocheon strain의 누에에 대한 병원성에 차이를 보이지 않았다.

The four genetically distinct isolates have been identified previously from Bombyx mori nucleopolyhedroviruses (BmNPVs) isolated in Korea. To further understand the complex of viruses infecting Bombyx mori, the genome of BmNPV-K1 and K4 strains was completely sequenced and analyzed in comparison with the genome of other sequenced baculoviruses including previously reported BmNPV. BmNPV-K1 consisted of 127,542 bp and 133 open reading frames (ORFs) of 150 nucleotides or longer with minimal overlap have been identified. In contrast, BmNPV-K4 consisted of 128,615 bp and 134 open reading frames (ORFs). Although gene arrangement is virtually identical, the genome of BmNPV-K4 is 1,073 bp longer than BmNPV-K1. This was related to the more existence of bro genes in BmNPV-K4. To investigate the relationship between BmNPV-K1 and K4, phylogenetic analysis with each member of the paired ORFs was performed. The sequence data suggest that BmNPVK1 and BmNPV-K4 are closely related but have diverged and evolved into two separate strains. This was study to identify highly related but separately evolving viruses in the same insect host and geographic location. We are currently comparing the differences of these BmNPV genomes to elucidate characteristics of each virus.

The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP4, GP5, and M. They have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. However, the PRRSV structural proteins are produced in low levels in the infected cells because it forms insoluble protein and possesses several transmembrane regions. To overcome this problem, we fused the GP4, GP5, and M with SUMO (Small ubiquitin-related modifier), and expressed the fused gene in Bm5 cells and silkworm larvae. Expression of the proteins were analyzed by 12% SDS-PAGE and western blotting using 6xHis tag and porcine anti-PRRSV antibodies. In results, SUMO fused proteins were expressed at a high level in Bm5 cells. The levels of protein using the silkworm larvae is higher than that using Bm5 cells. The fused protein was purified by Ni-NTA affinity chromatography. This study demonstrated that SUMO, when fused with PRRSV structural proteins, was able to promote its soluble expression. This may be a better method to produce PRRSV structural proteins for vaccine development.

For stable germline transformation, the promoter of B. mori cytoplasmic actin gene (BmA3) was used to ubiquitous expression of transgenes. Except for BmA3 promoter, promoters used to regulate gene expressionin all tissues and developmental stages of B. mori were not nearly developed. To identify more powerful promoter than previously reported BmA3 promoter (Mange et al., 1997), we introduced a new dot blot hybridization method, and isolated nine clones that show stronger dot signal compared to the control, BmA3by this method. Among these 9 clones, we focused on one clone which has high amino acid homology (94%) with heat shock protein 70 gene of Trichoplusia ni. This resulting positive clone, named bHsp70 (B. mori heat shock protein 70) was ubiquitiously expressed in tissues and developmental stage of fifth instar B. mori larvae,and stimulated bythermal and ER stress. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1003/+147) in the 5'-flanking region of bHsp70 gene that has 264-fold more intensive promoter activity than BmA3 promoter. Moreover, transcription activity of bHsp70 promoter under heat shock condition (42 ℃, 4 hr) was increased over 2-fold than normal condition. Therefore, we suggest that bHsp70 promoter may be used more effective candidate for transgene expression in B. mori.

The silkworm (Bombyx mori), as an industrial insect, possesses a high economic value. Casual discrimination and accumulated genetic information of silkworm varieties are essential ground for the practical utilization and long-term conservation. In this study, nine available microsatellite loci were successfully genotyped from ~50 silkworm strains preserved in Korea. According to genotyping analysis, we obtained 3 ~ 16 alleles per locus, with an average of 7.4, the observed heterozygosity ranging from 0.04 to 0.98, and the polymorphic information content (PIC) ranging from 0.06 to 0.88, revealing that some loci are highly variable. Among 54 strains 13 strains were casually identified by the presence of 17 strain-specific apomorphic alleles. Furthermore, 30 among remaining strains contained strain-specific allele combinations that are also apomorphic to each strain, allowing us to discriminate each of these from other strains by genotyping of multiple loci. These results collectively suggest that the silkworm microsatellite DNA is actually and potentially important molecular marker for the discrimination of the silkworm strains that are preserved as hundreds in Korea, as more loci are genotyped.

MicroRNAs (miRNAs) are endogenous non-coding genes that participate in post-transcription regulation by either degrading mRNA or blocking its translation. It is considered to be very important in regulating insect development and metamorphosis. Insects are the largest group of animals and are extremely valuable in biological and agriculture research. Insects are also important pests to human health and agriculture, and efforts are necessary protect both humans and plants from disease and damage. Despite their importance, insects lag behind mammals, nematodes, and plants in miRNA research. At present, only 279 insect miRNAs have been identified from Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Bombyx mori, and D. pseudoobscura in miRBase, and most of these miRNAs were computationally predicted without experimental validation. Functional analysis of insect miRNAs has only been conducted in D. melanogaster.

For the purposes of this paper, stress may be defined as any modification of environmental parameters that leads to a response by biological organisms. Stresses that affect biological structures may be non-thermal, such as ultraviolet radiation, pH, or salinity, or thermal. Temperature is one of the major stresses that all living organism face. The major effects of cold shock are decrease of membrane fluidity and the stabilization of secondary structures of RNA and DNA in the cells, which may effect the efficiency of translation, transcription, and DNA replication. In this study in compliance with the cold temperature stress about selection of the useful gene is contents from the silkworm which is been revealed. The survival rate which is caused by with the cold temperature stress until 12 hours was 100% in 0℃, until 2 hours was 100% in -5℃. A total of 960 clones were randomly selected from the subtraction cDNA library, and then performed a differential display hybridization analysis with forward and reverse probes. In conclusion, selected 53 partial clones and novel 2 full-length clones were identified as differential expressed genes. We assumed that the novel gene related with transmembrane.

Background: Proteolytic enzymes are involved in insect molting and metamorphosis and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compared the expression profile of B. mori cathepsins B (BmCatB) and D (BmCatD) in the fat body during the larval-pupal transformation of B. mori in the BmCatB or BmCatD RNA interference (RNAi) process. Results: BmCatB is ecdysone-induced and expressed in the fat body of B. mori during the molting, and the larval-pupal and pupal-adult transformations, and its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval-pupal transformation and BmCatB RNAi treatment resulted in the arrest of the larval-pupal transformation. RNAi-treated BmCatB knock-down sustained the expression of BmCatD during the larval-pupal transformation. On the other hand, BmCatD RNAi up-regulated the expression of BmCatB in the fat body of final instar larvae. Conclusion: Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis and that BmCatB and BmCatD contribute collaboratively to B. mori metamorphosis

Disulfide bond formation, reduction and isomerization are important posttranslational modification in proteins that occur in most, if not all, living organisms. In eukatoyes, disulfide bond in substrate proteins are primarily formes by ERO1 and PDI. ERO1, oxidized by molecular oxygen, acts as a specific oxidant of PDI, which then makes disulfide bonds in folding proteins oxidized directly. It means that ERO1 plays an essential role in setting the redox potential in the ER, and the regulation of Ero1p activity is critical to maintain redox homeostasis and proper ER folding activity. We have isolated and analysed a endoplasmic reticulum oxidoreductase (ERO1) from Bombye mori. It apperas that both an N-terminal CxxxxC motif and a C-terminl CxxCxxC motif are necessary for Ero1p fuction. In vivo, the result of the 5day of 5th instar larvae by RT-PCR and Real-Time PCR shows that posterior silkgland, skin and mid silkgland are revealed more than rhose of other tissues. The same result for tissue distribution of transcripts is appeared about ERO1 and PDI. In Bombyx mori, ERO1 is also supposed to correlate with PDI. Afterwards, more experiments are needed to figure out accurate interrelation between ERO1 and PDI.

Recently Transgenesis was achieved in Bombix mori. For stable and effective transgenesis in B.mori, B.mori cytoplasmic actin gene (BmA3) promoter was used to expression of marker gene, the green fluorescent protein(GFP). Green fluorescent protein expression for selection of transformants was visible in all larval, pupal, and adult tissues but, unexpectdly, was not detectable in embryos. So, it spend times and money on rearing of silkworm. Furthermore, the BmA3 promoter is predominantly active in the midgut, which makes it difficult to reliably identify transformants since autofluorescence of many insect foods can mask low-level fluorescence and only allows the detection of strongly expressing individuals with potentially multiple insertions. Therefore, we need more intensely promoter than BmA3 promoter for selected by expression of GFP in embryos and selected by reliable expression of GFP in larvae. We performed dot blot hybridization to develop strong promoter. Nine differentially expressed clones were isolated and we focused one clone of them which has high similarity with heat shock protein 70 gene from D.melanogaster. We named it as bHSP70 (Bombyx mori heat shock protein 70). Expression from the hsp70 promoter was strong and heat shock-dependent. And Drosophila hsp70 promoter appears useful for regulating expression of Exogenous DNA. So, we analyzed transcriptional activity of promoter with bHSP70 gene by using dual luciferase assay system. bHSP70 promoter has about 264 folds more intensely than BmA3 promoter. Also, when bHSP70 promoter treated heat shock(42℃), transcriptional activity incresed 2 times more than normal condition. Therefore, we suggest that bHSP70 promoter is more effective candidate for stable transformation and selection of transformants.

Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the slow cis/trans isomeraization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. We studied the characterization of Cyclophilin A (bCyp A) isolated from Bombyx mori . The cDNA of bCyp A is 947 bp. There is a 5´-untranslated region of 91 nucleotides followed by an initiating ATG codon. The TAA termination codon occurs at nucleotide 588. Thus translation of the sequence from nucleotides 91 to 588 would produce a protein of 166 amino acids with a calculated molecular mass of 18.2kDa. The 'AATAAA' consensus polyadenylation signal and poly A tail are present in the 3´-untranslated region. To analysis of PPIase activity, we expressed the bCyp A protein in Sf9 cell by using baculovirus expression vector system (BEVS). SDS-PAGE and Western blot analysis showed that the molecular weights of intracelluar expressed protein was approximately 28.2 kDa. The PPIase activity assay was monitored by proteolytic cleavage of the chromophore p-nitroanilide byα-chymotrypsin. As substrate the synthetic tetrapeptide succinyl-Ala-Ala- Pro-Phe-p-nitroanilide was used.

Electroporation is well known today as a powerful transfection technique and is useful for the study of gene expression. The advantage of the electroporation method is that large quantity of silkworm (Bombyx mori) eggs can be transformed in a very short time. However, how to use it for introducing foreign gene into silkworm eggs needs systematical investigation. In our silkworm transgenesis program, we needed an efficient technique to evaluate the functionality of transgenes before their injection into eggs. The goal of this experiment was to find an alternative efficient method of generating transgenic silkworm eggs using a commercially available electroporation device. The Gene Pulser Xcell (Bio-Rad Laboratories, USA) were used. In contrast to other electroporation devices, which are based on a single pulse with exponential decay or square wave technology. We investigated pigmentation-rate and hatching-rate of the silkworm eggs of electroporation. We used foreign gene LacZ, EGFP, Ds-red induced vector system with selection marker for transgenic silkworm. The LacZ gene in 3rd instar larva DNA can be detected by β-galactosidase stain. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is available technique for transfecting B. mori egg.